Aldoheptose Biosynthesis. An E. coli K-12 strain carrying a cysE-pyrE linked mutation, designated rfaD, which affects the synthesis of L-glycero-D-mannoheptose was isolated and genetically characterized. The rfad phenotype includes, in addition to altered LPS synthesis, increased permeability to a large number of hydrophobic antibiotics. The rfad gene initially identified in the Clarke-Carbon Genomic Bank was cloned in pBR322, and, subsequently, smaller restriction fragments were cloned into several plasmid vectors. The precise location of the rfad gene on a 1.3-kilobase SspI-HpaI fragment has been determined. The x-fad gene and the flanking regions have been completely sequenced, and the coding and regulatory regions have been defined. The location of the rfaD gene on the physical map of the Escherichia coli chromosome has been resolved. RfaD plasmids express in vivo and in vitro a protein with the molecular weight of 37,000. The protein, ADP-L-glycero-D-mannoheptose-6-epimerase, has been purified to homogeneity and partially characterized. N-Terminal analysis of purified ADP-L-glycero-D-mannoheptose-6-epimerase confirms the first 34 amino acid sequence deduced from the nucleotide sequence of the rfad gene coding region. The primary structure of the rfad protein contains the sequence fingerprint for the ADP-binding beta-alpha-beta-fold at the N-terminus.